Obiettivo
The central objective of this programme of work has been the structural elucidation of the functional domains of PtdIns-specific phospholipase C-beta2 (PtdIns PLCbeta2).
Much of this effort has been directed at a reduction in complexity of the problem through definition of subdomains for nmr-based structural elucidation. This has been proven to be an intractable problem for some of the protein domains either due to size or to solubility. This has led the network to their specified contingency plan with further work directed towards crystallographic solutions for the protein.
The amino-terminal domain of PtdIns PLCbetab2 has been defined as the target site for Gprotein betagamma interaction. Mutagenesis of this region reveals that subdomain 4 and 5 of this putative PH domain are important for betagamma binding. This domain also interacts with substrate and InsP3 analogous to PLCdelta1. Large scale production of the PLCbeta2 PH domain has been achieved in bacteria although solubility has proven a major problem. A refolding protocol has been developed for this domain and sufficient quantities of soluble protein are now produced. Preliminary nmr analysis demonstrates that the protein is folded and this now provides the basis for the full structural determination.
Synthetic constrained oligopeptides designed to modulate function (inhibit Gbetagamma-activation) were based upon the predicted 'PH-domain' and mutagenesis. Final products were fully characterised and tested. Contrary to expectation, Gbetagamma-independent activating responses were observed. While this may infer second-site interactions with the catalytic core domain, only structural solution will resolve this.
The bacterial expression of a functional catalytic domain of PtdIns PLCbeta2 has proven to be impossible. While success was achieved for the PtdIns PLCdelta1 expression yielding a full structural solution, the information produced on domain boundaries for the equivalent PtdIns PLCbeta2 constructs has not led to the production of soluble active protein. Thus sequential aminoterminal deletion constructs, varying growth and induction conditions has not produced suitable bacterial expression. In order to make progress on this structure, the problem was switched to eukaryotic expression. Baculovirus expression has thus been employed to produce tens of milligrams of a C-terminally truncated PtdIns PLCbeta2 (it has been necessary to remove this C-terminus because proteolysis in this region creates heterogeneity of the purified PtdIns PLCbeta2). This protein has been crystallised albeit in a form that diffracts poorly. The improvement of these crystals is ongoing and alternative crystallisation conditions also are being sought in a continuing effort.
The C-terminal regions of PtdIns PLCbeta2 has been subdivided into two domains based upon proteolysis. These have been shown to be separated by a region subject to protein kinase C phosphorylation and evidence indicates the regulated 'exposure' of this region. The protease-resistant C-terminal domain is too large for nmr and has been produced in tens of mg amounts in E.Coli for crystallography; conditions are being screened for crystallisation. The N-terminal part of this domain while of a size suitable for nmr analysis has proven to be entirely insoluble under all conditions. Solution of this region is being sought through bacterial expression of the entire C-terminus, which has also been produced in mg quantities in E.Coli in a soluble form. Crystalisation conditions are being screened for this domain.
In summary, the properties of PLCbeta2 subdomains has forced the network towards crystallographic structural solutions in all but one case. Nevertheless, the programme has provided structural coverage of the entire PtdIns PLCbeta2 protein and current work should resolve the complete structure. The time limit placed upon the study makes it inevitable that these structures while facilitated by the Lipase programme will not be completed until beyond the expiry of EC support.
Campo scientifico (EuroSciVoc)
CORDIS classifica i progetti con EuroSciVoc, una tassonomia multilingue dei campi scientifici, attraverso un processo semi-automatico basato su tecniche NLP. Cfr.: Il Vocabolario Scientifico Europeo.
CORDIS classifica i progetti con EuroSciVoc, una tassonomia multilingue dei campi scientifici, attraverso un processo semi-automatico basato su tecniche NLP. Cfr.: Il Vocabolario Scientifico Europeo.
- scienze naturali scienze biologiche microbiologia batteriologia
- scienze naturali scienze della terra e scienze ambientali connesse geologia mineralogia cristallografia
- scienze naturali scienze biologiche biochimica biomolecole proteine
- scienze naturali scienze biologiche biochimica biomolecole lipidi
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Coordinatore
WC2A 3PX London
Regno Unito
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